[Establishment and application of a novel method for detecting MLL fusion genes of acute leukemia].

This study was aimed to establish an efficient method to detect 10 common MLL fusion genes in patients with acute leukemia. Firstly, the relevant references and databases were searched to thoroughly investigate all fusion breakpoints; the primers and probes were designed according to nearly all the involved fusion types of gene. Then the multiplex real-time PCR system was established and optimized by using the established 16 positive plasmids and negative cell lines.

[A rapid screening program on the resistance to streptomycin and ethambutol in Mycobacterium tuberculosis isolates by PCR melting curve analysis].

Objective: To evaluate the effects of PCR melting curve analysis assay on a rapid screening program regarding the resistance of Mycobacterium tuberculosis (MTB) clinical isolates to streptomycin and ethambutol.

Methods: A total of 331 clinical isolates of MTB had been collected since 2007-2009 in Shenzhen. Mutations at codon 306, 378-380, 406 and 497 of embB gene, codon 43, 88 of rpsL gene, and 513-517, 905-908 region of rrs gene were detected by PCR melting curve analysis.

[Rapid detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis using real-time PCR and melting curve analysis].

Objective: To evaluate the application of a real-time PCR and melting curve analysis assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis (M. tuberculosis).

Methods: A total of 311 clinical isolates of M.

[Evaluation of methods for detection of NPM1 gene mutations in acute myeloid leukemia].

The purpose of this study was to establish real-time based methods for detection of NPM1 gene mutation in acute myeloid leukemia (AML). Primers/probes were designed according to the clustered region of NPM1 mutations on exon 12. Two real-time PCR assays, including high resolution melting curve (HRM) and allele-specific PCR (AS-PCR), were developed and clinically evaluated with 89 AML samples, which were parallelly detected by capillary electrophoresis (CE) and sequencing.

[Analyzing the mutations of rpoB gene in Mycobacterium tuberculosis clinical isolates by probe melting analysis assay].

Objective: To evaluate the clinical performance of a probe melting analysis (PMA)-based real-time PCR detection kit in rapid detection of rifampin-resistant mutations in Mycobacterium tuberculosis (MTB).

Methods: The specificity of the assay was evaluated by detecting 37 non-tuberculous mycobacteria (NTM), and the detection limit of the method was evaluated by genomic DNA of a standard strain H37Rv. Finally, 962 clinical isolates were analyzed with the PMA assay by detecting mutations in rifampin resistance-determining region (RRDR) of rpoB gene, and results were verified with DNA sequencing.

[Application study on multicolor combinational probe coding real-time PCR in detection of foodborne pathogens].

Objective: To investigate the detection limit of multicolor combinational probe coding real-time PCR (MCPC-PCR) in detection of Salmonella and Staphylococcus aureus suspended in the food samples, and to apply MCPC-PCR to detect the samples of food poisoning.

Methods: Series concentration of bacterium suspension (10(1) - 10(9) CFU/ml) was prepared by using 22 simulated samples including fresh meat and cakes and then MCPC-PCR was applied to detect Salmonella and Staphylococcus aureus in 22 samples. Enrichment broth of 101 frozen samples and 5 early patients' anal swabs in food poisoning cases were detected after the DNA samples were extracted.

[Rapid simultaneous detection of Salmonella and Shigella using modified molecular beacons and real-time PCR].

Objective: Dual detection of Salmonella and Shigella using modified molecular beacons and real-time PCR was developed. The established method was applied to rapid diagnosis of Salmonella and Shigella' food poisoning, and for routine monitoring programs.

Methods: Two sets of primers were designed based on the core sequence of invA gene and ssaR gene published on GenBank to detect Salmonella, and ipaH gene were selected to detect Shigella.

[Detection of APC mutation by real-time polymerase chain reaction and melting curve analysis].

Objective: To establish a simple, reliable and low cost approach for clinical detection of APC mutation.

Methods: Using SYBR Green I as the real-time polymerase chain reaction (PCR) product indicator, a DNA fragment of 270 bp targeting APC_1309 mutation (5 bp deletion) was amplified from the sample DNA. A short fragment (40/35 bp) was then amplified from the 270 bp PCR product, followed by melting curve analysis from 65 degrees C to 99 degrees C at 0.

Quantitative detection of a marine fish iridovirus isolated from large yellow croaker, Pseudosciaena crocea, using a molecular beacon.

A real-time polymerase chain reaction (PCR) assay utilizing a molecular beacon for the quantitative detection of a marine fish iridovirus isolated from large yellow croaker, Pseudosciaena crocea (LYCIV), was developed, which involved the amplification of a 122bp DNA fragment from a conserved region of LYCIV ATPase gene. The specific probe consisting of two short arm and a central loop sequences complementary to the target amplicon was characterized with respect to its efficiency of quenching (E(ff)), and signal to background ratio by spectrofluorometric analysis of its hybridization with the complementary oligonucleotide target. The positive control plasmid pFHT-ATPase containing the target sequence was quantified to make the standard curve for sample detection after serial 10-fold dilution.

[Construction of RNA-containing virus-like nanoparticles expression vector with cysteine residues on surface and fluorescent decoration].

Site-directed mutagenesis was performed at the codon 15 of the MS2 bacteriophage coat protein gene,which had been cloned to the virus-like particles expression vector containing non-self RNA fragment. The produced expression vector,termed pARSC, was transformed to E. coli DH5alpha.

[Screening C282Y mutation with double-stranded probes using synchronous fluorometry].

We described in the paper a new high-throughput screening method for Cys282Tyr mutation in hereditary haemochromatosis with double-stranded probe using synchronous fluorometry. The probe for wild type was labeled with Fam, the probe for mutant type was labeled with Joe. After PCR, reaction tubes were transferred to a spectrofluorometer, where synchronous spectra were scanned in a constant-wavelength mode.